Subject matter of the invention is a method of detecting a substance to be analyzed in a sample using a probe which contains nucleobases.
The detection of low-concentration substances to be analyzed in samples has become very important in clinical testing. As a result of numerous developments, the detection limit for substances to be analyzed has been lowered considerably compared with earlier tests. This development has had a particularly strong impact in the field of nucleic acid detection. Many of the detection systems used today, however, involve complex working steps or use reagents that are very complex to manufacture. These systems have many disadvantages.
In U.S. Pat. No. 4,683,202, a method for detecting nucleic acids is described that is based on the amplification of segments of an original nucleic acid. This method has become known as the polymerase chain reaction. A disadvantage of this method, however, is that the results are difficult to quantify.
Results are easier to quantify when nucleic acid probes are used with which stoichiometric hybridization takes place. Unfortunately these methods are insensitive.
In a further development of the traditional hybridization test, it was therefore proposed that a number of identical nucleotide sequences be bound with each probe. The hybrid consisting of analyte nucleic acid and probe was detected by hybridizing a number of secondary probes that were complementary to these identical nucleotide sequences. A number of documents that describe the prior art discuss the most favorable arrangement of the many identical nucleotide sequences in the probe or in a set consisting of probes that are capable of hybridizingxe2x80x94and, therefore, aggregatingxe2x80x94with each other (e.g., U.S. Pat. No. 5,424,188; EP-B-0 248 896 B1; U.S. Pat. No. 5,424,413; 5,437,977). It is stated in U.S. Pat. No. 5,424,188, for instance, that the identical nucleotide sequences can be attached to each other in linear fashion. The chain of identical nucleotide sequences produced can also be formed into a closed ring. It is stated in U.S. Pat. No. 5,124,246 that the identical nucleotide sequences can be bound to each other by means of a branched structure. A distinction is made in this case between fork-like branchings and comb-like branchings. A number of potential technical embodiments have been described for the branchings (Nucleic Acids Research 16/11, 4937-4965, Gene 61, 253-264, Nucleic Acids Research 17/17, 6959-6967, Clinical Chemistry 35/8, 1571-1575 (1989), Bioorganic and Medicinal Chemistry Letters 4/8, 1011-1018 (1994), Nucleic Acids Research Symposium 24, 197-200 (1991), and Clin. Chem. 39/4, 725-726 (1993)). In WO 95/01365, a branching method is also described in which the lateral arms are bound to a special backbone by means of a reaction between phosphorothioates and haloacyl groups. Methods in which the probe has a number of identical nucleotide sequences for hybridizing with secondary probes can often lead to increased calibration curve sensitivity. The probes required for this, however, are 1) difficult to manufacture, and 2) the labelled probes do not stoichiometrically saturate the nucleic acid sequences on the probes. The test sensitivity achieved is therefore much less than that of the PCR method (AIDS 7/suppl., 11-14 (1993); J. Med. Virol. 43, 262-268 (1994); J. Infect. Dis. 170, 1172-1179 (1994); AIDS Res. and Human Retrovir. 11/3, 353-361 (1995)).
The one feature that all of these concepts have in common is that at least two different types of probesxe2x80x94one of which is present in great numbersxe2x80x94are aggregated into one detectable amplification unit based on the principle of hybridization of single-stranded overhangs.
Object of the invention was, therefore, to improve the prior art and, in particular, to develop a sensitive method of detection that can be quantified directly.
Subject matter of the invention is therefore a method for detecting a substance to be analyzed in a sample by contacting the sample with a probe which contains nucleobases and two or more non-nucleosidic, label-attracting groups under conditions in which the substance to be analyzed binds indirectly or directly to the probe and detecting the binding product.
A substance to be analyzed according to the invention can be any ingredient in a sample. Preferably, however, the substance to be analyzed is a molecule that can be detected immunologically or by means of base pairing. Immunologically detectable substances to be analyzed are, for instance, antibodies, antigens or haptens. Nucleotides are substances to be analyzed that can be detected by means of base pairing. They include ribonucleic acids and deoxyribonucleic acids. These types of substances to be analyzed can be detected in practically any sample using the proposed method. In many cases, however, it is preferable to pretreat the sample so that the substance to be analyzed is present in a form that facilitates detection. This includes releasing the substance to be analyzed from compartments, e.g., cells, in which the analyte was originally enclosed. The substance to be analyzed can itself be a compartment, however, if it has detectable components on its surface, such as surface antigens. In addition, the actual substance to be analyzed can be amplified before the method provided by this invention is carried out, e.g., using the PCR, but preferably only to the degree that the amplification can still be quantified. Blood, urine, sputum or swabs have proven to be suitable fluids from which a sample suitable for detection can be produced. Another possible preparation step is to liquify viscous samples or to partially purify the analyte, e.g., by immobilizing it on a solid phase. The analyte can be present in a liquid, e.g., in dissolved or suspended form. The analyte can also be bound to a solid phase, however. Suitable solid phases include latex particles, magnetic particles, or vessel walls.
A probe according to the present invention is a molecule that has a first nucleic acid-type portion, and a second portion that is analyte-specific or that promotes contact with the analyte. The nucleic acid-type portion contains nucleobases on a backbone. Nucleobases include the naturally occurring bases A, G, C, T, and U, or any bases derived therefrom. A potential backbone is the natural sugar phosphate structure. Molecules that have a polyamide backbone were also described recently (e.g., in WO 91/20702). The probe can bind the analyte directly or indirectly by means of the analyte-specific portion. In cases of direct binding, the nature of the analyte-specific portion can depend on the type of substance to be analyzed. If the substance to be analyzed is immunologically active, binding can take place with the partner that is immunologically complementary to the analyte. To detect an antigen, for instance, a probe can therefore be used that contains an antibody to this antigen that is bound covalently or non-covalently to the nucleic acid-type portion. If the substance to be analyzed is a nucleic acid, the probe contains a nucleobase sequence that is complementary to one part of the base sequence of the analyte. An indirect binding of the probe to the analyte can be achieved by directing the analyte-specific portion against a part of a promotor probe that can bind with the analyte. The promoter probe therefore preferably contains an analyte-specific portion and a portion that is capable of binding with the analyte-specific portion. In this case it is preferable for the probe and the promoter probe to bind with each other by means of base pairing. In this case, a promoter probe is preferably selected that has a portion that is complementary to the analyte and a portion that is complementary to the probe. The advantage of this method is that a number of relatively simply constructed promoter probes be used that correspond to the number of analytes to be detected in order to detect various analytes, but only requires one relatively complex probe.
The nucleic acid-type portion of the probe is designed in such a way that it cannot form hybrids with other nucleic acids found in the reaction mixture or sample mixture. The nucleic acid-type portion can be linear. The nucleic acid-type portion is usually sterically complex, however, and a preferred embodiment is branched many times within itself. This portion is preferably branched in chemically covalent fashion and, especially preferred, by means of functional groups between (deoxy-)ribose units. The portion also preferably contains a number of identical nucleotide sequences. The manufacture of branched molecules of this nature is described in detail in the prior art described earlier. A few concepts for manufacturing branched, nucleic acid-like portions are explained below.
According to one embodiment, nucleic acids that are made by inserting N4-(N-(6-trifluoroacetyl-amidocaproyl)-2-aminoethyl)-2xe2x80x2-deoxycytidine during phosphoramidite synthesis are cross-linked using p-phenylene diisothyocyanate (DITC) as a homo-bifunctional linker, and a suitable, double-stranded fraction is isolated from the product mixture, e.g., star-structured multimers. The manufacture of these multimers is described in xe2x80x9cLuminescence Immunoassay and Molecular Applicationsxe2x80x9d (1990, Editor: Knox Van Dyke, CRC Press, Boca Raton, USA). Another possibility for branching is provided by branching monomers that have a total of three hydroxyl groups per nucleotide. The additional hydroxyl group that is orthogonal to the 5xe2x80x2, 3xe2x80x2-axis can be bound with cytidine in position N4 by means of a hexamethylene group, for instance. Both the 5xe2x80x2-hydroxyl group and the hydroxyl group located on the base (which formerly had the same protective group) react with a nucleoside phosphoramidite during oligonucleotide synthesis according to phosphoramidite procedure. As a result, the oligonucleotide chain being constructed branches every time a branching monomer is used. By varying the protective groups, a linear DNA strand can be synthesized that has branching points everywhere that these branching monomers were incorporated. Subsequent selective cleavage of the protective group of the second hydroxyl group enables the lateral strands to be synthesized separately. These secondary sequences project from the primary sequence like teeth in a comb (comb structures). These methods are described, for instance, in Nucleic Acids Research 17/17, 6959-6967 (1989) and Nucleic Acids Research Symposium 24, 197-200 (1991).
A core of the invention is the fact that the probe has two or more label-attracting, non-nucleosidic groups within its nucleic acid-like portion. The invention therefore differs from the prior art in that the long, identical nucleotide sequences xe2x80x94which have been used previously as label promotersxe2x80x94are replaced with low molecular, relatively small, non-nucleosidic groups that are incorporated in comparatively close intervals (and are preferably spacered in flexibly away from the backbone). These label-attracting groups are sterically not very complex. Preferable label-attracting groups are relatively small, immunologically detectable groups such as haptens. Especially preferred in terms of the invention are well-soluble haptens that represent a strong antigenic determinant. Candidates include digoxigenin (U.S. Pat. No. 5,344,757), nitrophenols (such as dinitrophenol or nitro-iodophenol), and especially sugars, e.g., lactosamine, or suitable pharmaceuticals. Other examples include the 4-hydroxyl-3-nitro-phenacetyl group (NP), e.g., coupled to an amino spacer such as amino capronic acid as carboxamide via EDC, and further activated by esterification with NHS to form (N-hydroxysuccinimidyl)-xcex5-aminocaproyl-NP-carboxamide (this enables aminoalkyl-derived phosphonate bridges, nucleic bases or sugars to be attacked, for instance), the 4-hydroxy-3-iodo-5-nitro-phenacetyl group (NIP), e.g. coupled to an amino spacer such as amino capronic acid as a carboxamide via EDC, and further activated by means of esterification with NHS to form (N-hydroxysuccinimidyl)-xcex5-amino caproyl-NIP-carboxamide (this enables aminoalkyl-derived phosphonate bridges, nucleic bases or sugars to be attacked; specific  less than NIP greater than -antibodies are described) and 1-dimethoxy trityl-3-O-(N-(2,4-dinitrophenyl)-3-aminopropyl)-triethylene glycol)-glyceryl-2-O-(cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (DNP-TEG) (DNP-TEG can be inserted directly using the phosphite method during DNA synthesis; specific  less than DNP greater than -antibodies are described).
The label-attracting groups can be inserted into the nucleic acid-type portion after it is synthesized. This can be accomplished, for instance, by reacting the nucleic acid-specific portion with compounds that are capable of bonding covalently with groups of nucleic acids. These include reagents in particular that can react with functional groups attached to nucleic acids, e.g., exocyclic amino groups of bases. A suitable method is described in EP-A-0 173 251, for instance. It is also possible to manufacture the label-attracting group by photocoupling the nucleic acids with a reagent that can be photoactivated. A method of this nature is described in U.S. Pat. No. 5,344,757, for instance. This patent describes a label using a digoxigenin group.
It is also possible, however, to use the label-attracting group in the form bound to mononucleotide components that was already used during synthesis of the nucleic acid-type portion. Phosphoramidites can be used in phosphoramidite synthesis, for instance, in which label-attracting groups or protected precursor groups are bound to exocyclic amino groups of a base or to the internucleoside phosphodiesters that are formed.
The probes according to the invention are preferably single-stranded, chemically synthesized compounds.
Another possible method of manufacturing is based on the synthesis method described in WO 95/01365. It is based on the very efficient coupling of thiophosphate or dithiophosphate groups and haloacyl or haloalkyl groups. Thiophosphate and betabromoacetamide functions that couple and form thiophosphorylacetamide bridges are preferably used. Branched multimers that can be used as the nucleic acid-type portion can be made as follows. In the first step, an initial oligonucleotide that is amino or aminoalkyl-derivatized on the 5xe2x80x2-end is reacted with N-hydroxysuccinimedyl-beta-bromo-acetate, forming a terminal beta-bromoacetamido function. In the second step, a second oligonucleotide is functionalized as 3xe2x80x2-O-phosphoryl-(2-aminomethyl-) ethyl-thiophosphate ester on the 3xe2x80x2end. The terminal thiophosphate group of the second oligonucleotide then reacts, with a nucleophilic attack on the bromo-substituted beta-C atom on the 5xe2x80x2-end of the first oligonucleotide, and both oligonucleotides are ligated. The aminomethyl function is then activated into a beta-bromoacetamido function via condensation with N-hydroxy succinimidyl-beta-bromoacet ate.
Larger pieces of oligonucleotides can be manufactured by performing corresponding functionalization steps on the 3xe2x80x2and 5xe2x80x2-ends of numerous oligonucleotides and performing the steps described above. These oligonucleotide pieces have many functionalized beta-bromoacetamido groups, which represent potential branching points. Activatable amino groups can also be inserted directly into the oligonucleotide strand using amino alkyl phosphite derivatives during phosphoramidite synthesis. The advantage of this method is that the primary sequence with potential branching points can be thoroughly synthesized on a CPG carrier. In a subsequent step, a third oligonucleotidexe2x80x94which was manufactured by inserting nucleotides that were functionalized using a label-attracting groupxe2x80x94is converted to thiophosphate ester on the 5xe2x80x2-end. Branched multimers can now be made directly (without templates) by reacting this oligonucleotide with the inserted beta-bromoacetamido functions. These branched multimers represent the basis for the desired signal amplification based on the number of branching points and detectable labels per branching. Unlike the method described in WO 95/01365, relatively short third oligonucleotides can be inserted in the method according to this invention. As described above, they are preferably already labelled with a label-attracting group when attached to the branching points, e.g., when they are synthesized by means of the phosphoramidite method (e.g., per EP-A-0 399 330).
The label-attracting group is preferably detected indirectly. It is preferably detected using a conjugate of a group displaying affinity with the label-attracting group, and a signal-producing component. Such groups displaying affinity are groups, for instance, that react immunologically with the label-attracting group and bind the conjugate to the label-attracting group. In the case of haptens, antibodies directed against this hapten can therefore be used as groups displaying affinity. When the hapten is digoxigenin, antibodies against digoxigenin are available to form conjugates.
Signal-producing components according to the invention are groups that can either be detected directly orxe2x80x94preferablyxe2x80x94transformed into a detectable component in a chemical reaction. Especially preferred signal-producing components are relatively small proteins, especially the calcium-activatable photoproteins. A family of molecular systems capable of producing luminescence falls under the concept Ca2xe2x88x92-activatable photoproteins. They share the following characteristics:
a. Physico-chemical aspects
a reaction complex in final form consisting of a proteinaceous catalyst (=apo-photoprotein), an organic-chemical substratexe2x80x94which is firmly yet non-covalently bound, and which represents the actual emitter (=luminescent substance)xe2x80x94and molecular oxygen, which is also firmly fixed (probably covalently protein-bound as hydroperoxide). The molecular oxygen is required to initiate the luminescence reaction (=oxidant)
differs from enzyme systems in that all components required for the luminescent reaction are bound
has a relative molecular mass of about 22,000 daltons
xe2x80x9ccoelenterazinexe2x80x9d ([2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-8-benzyl-7-hydroi-imidazopyrazine-3-on] is the luminescent substance
emission peak wavelength (xcexc max) is in the blue range (approx. 470 nm)
the luminescent reaction is initiated by the binding of 2-3 Ca2+ ions to the apo-photoprotein
Ca2+ binding domains are present in the apo-photoprotein in the form of EF hand (=helix-loop-helix) structures
luminescent reaction does not depend on ambient oxygen, i.e., the complete photoprotein luminesces in the presence or absence of O2 in the surrounding atmosphere
photoprotein reaction takes place in the form of flash kinetics, i.e., a concentrated light emission results from the addition of Ca2+ ions (cf Blinks J. R., et al., Pharmacological Reviews 28/1, 1-93, 1976)
b. Molecular biological aspects
functional apo-photoproteins with a peptide length of 189-196 amino acids
high concordance between nucleobases and amino acide sequences ( greater than 60% homology) of the individual representatives from the family of Ca2+-activatable photoproteins, as illustrated clearly by the cloning of aequorin (Prasher D., et al., Biochemical and Biophysical Research Communications 126/3, 1259-1268, 1985; Prasher, D., et al., Methods in Enzymology 133, 288-299, 1986; Prasher D., et al., Biochemistry 26, 1326-1332, 1987; Cornier M., et al., Photochemistry and Photobiology 49/4, 509-512, 1989), Obelin (Illarionov Boris A., et al., Gene 153, 273-274, 1995), Clytin (Inouye S. and Tsuji F., FEBS 315/3, 343-346, 1993) and Mitrocomin (Fagan T. F., et al., FEBS, 333/3 301-305, 1993).
Of all members of this family of Ca2+-activatable photoproteins, aequorin has been used most frequently in recent years with receptor-ligand binding assays. Preferred proteins include, e.g., obelin, halistaurin (=mitrocomin), phiallidin (=clytin) or aequorin. These proteins all emit a light signal when they are activated, which makes it possible to determine their quantity or presence by measuring the light intensity. The use of such photoproteins in traditional tests, and the mechanism by which it leads to signal formation, is described, for instance, in Cornier, M. L. et al., Photochem. and Photobiol. 49/4, 509-512 (1989) or Smith, D. F. et al. in xe2x80x9cBioluminescence and Chemiluminescence: Current Status (P. Stanley and L. Krick, eds.), John Wiley and Sons, Chichester, U.K. 1991, 529-532. The advantages of these signal-producing components are a shorter measuring time, increased quantum yield compared with other luminescence labelling systems, and a reduced reagent-induced background, i.e., the technical detection range of the luminometer that can fully exploited, and a broader dynamic range of measurement. The relatively small spatial requirement of the calcium-activatable photoproteins and their cofactors greatly reduces the size of the probe complex created after the conjugate binds. In addition, these calcium-activatable photoproteins do not interact with DNA structures, as is the case with acridinium salts or various chromophores, for instance.
Surprisinglyxe2x80x94and contrary to the general opinionxe2x80x94the absence of enzymatically catalyzed signal multiplication with this method as compared with enzyme labelling is more than outweighed in the proposed invention by the combination of minimal non-specific binding of small aequorin conjugates, minimal chemical noise from the label and the trigger, high specific photoprotein activity, and a very precise measurement of aequorin light flashes at a high signavnoise ratio.
The signal-producing components and the components displaying affinity to the label-attracting group are preferably attached by means of a well-solvatable linker with a length of at least 4 and preferably at least 8 atoms. This means that the degree of conformational freedom is increased and there is less steric hindrance. In turn, this can mean that the basic binding reactions can take place more quickly. A core of the invention is the fact that the distance between adjacent signal-producing components bound to the probe can be very small with the present invention. This is achieved in particular when the nucleotide repeat units used in the prior art are replaced with a nucleoside derivative labelled with a label-attracting group. The distance between the midpoints of two adjacent label-attracting groups on one nucleotide strand is preferably smaller than 18 or 15 nucleotides, yet larger than 3 nucleotides, with the present invention.
The distance between branching points in chemically covalently branched multimeric molecules is preferably less than 7 nucleotides, but is preferably at least 1 nucleotide.
The label-attracting group is preferably attached by means of spacer, i.e., a bridge consisting of 4 or more atoms. In branched probes, i.e., probes that contain a central primary sequence with numerous branching points and peripheral secondary sequences attached to the branching points, the label-attracting groups can be inserted in either the primary or secondary sequence, or in both sequences.
The method according to the invention works especially well with nucleic acid tests, i.e., when the substance to be analyzed is a nucleic acid. The advantages of the method according to the invention are also obvious in very simple processes such as detecting molecules on a surface, e.g. streptavidin bound to the surface of a tube wall. In this case, the probe is an oligonucleotide that contains a biotin molecule as the analyte-specific portion. The oligonucleotide portion is the nucleic acid-type portion to which two or more label-attracting groups, e.g., digoxigenin, are bound. In this method, a solution that contains the probe in an excessive quantity over the biotin binding sites of streptavidin is filled into the tube with a streptavidin-coated surface. After an incubation step, the excess probe is removed and a conjugate of antibodies to digoxigenin and aequorin is incubated in the tube with the immobilized probe. Then an excess quantity of the conjugate is removed. The reagents required to determine the aequorin label are then added, a triggering step takes place, and the light flash is measured. The intensity of the flash indicates the relative quantity of aequorin and, therefore, of bound probe. This, in turn, indicates the quantity of streptavidin on the surface.
An advantage of the method according to the invention is that the probe can be much smaller than in methods using signal-promoting hybridization zones, yet still retain the same signal intensity. It should be pointed out that the solubility of branched nucleic acids decreases very strongly as size increases. The fact that the probe according to this invention can be much smaller than previous probes while retaining the same number of label-attracting groups results in a higher solubility orxe2x80x94if the molecular size and solubility remain the samexe2x80x94a higher labelling density. It has been shown that the binding ratio of conjugates to the probe can be improved considerably if non-nucleosidic hapten groups and relatively small signal-producing componentsxe2x80x94and, therefore, conjugates (preferably smaller than 100 KD)xe2x80x94are used, i.e., access to the multiple labels is improved. This is accomplished because less space is occupied than with enzyme (e.g., alkaline phosphatase, xcex2-galactosidase)-labelled detection oligomer probes, and there is a greater degree of conformational freedom. In other words, the proper spacing between hapten and backbone, and between anti-hapten components and signal-producing components of the conjugate results in a more flexible spacial orientation. This makes it easier to quantify the analysis. In addition, with a hybridization immunoassay of this naturexe2x80x94unlike a pure hybridization assayxe2x80x94the label and conjugate incubation takes place at relatively moderate temperatures, e.g., 37xc2x0 C., which translates into considerable advantages in terms of retaining the specific activity of the conjugate added. In a pure hybridization assay, on the other hand, the requirements for specificity (highest temperature possible) and label activity (low temperature) work against each other. Thermal destruction of conjugate function immediately reduces the level of analytical test sensitivity that can be achieved. The method according to the invention improves multi labelling of the probe. In addition, labels can also be inserted into the primary sequence of the backbone without a significant increase in molecular size using the method according to this invention, in contrast to signal-promoting hybridization zones. The method according to the invention is also suitable for use in therapy monitoring.